Effect of high tyrosine content on the determination of tryptophan in protein by the acidic ninhydrin method. Application to chicken ovoinhibitor

Colorimetric determination of tryptophan in intact proteins by the acidic ninhydrin method of Gaitonde & Dovey (1970) gives high apparent tryptophan contents for proteins having high tyrosine/tryptophan ratios. Correction for this interference by tyrosine can be achieved by plotting the ratio of observed to expected tryptophan content as a function of tyrosine/tryptophan ratio for proteins of known composition. The equation of the line is: [Formula: see text] Application of this correction to chicken ovoinhibitor, which contains 17 tyrosine residues per molecule, gave results that agree with tryptophan content determined by other methods.     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.     

Spectrophotometric determination of tryptophan and tyrosine in peptides and proteins based on new color reactions

A new spectrophotometric method for quantitative determination of tryptophan and tyrosine in peptides and proteins is described. It is based on two specific color reactions, the reaction of tryptophan with formaldehyde and the reaction of tyrosine and tryptophan with hydroxylamine and ceric cations. By combination of these two reactions both tyrosine and tryptophan can be determined simultaneously. Tyrosine and/or tryptophan bound in peptides and/or proteins react independently of the rest of the peptide or protein molecule. The method is simple, accurate, and sensitive. Hydrolysis is not necessary.     

Quantitative determination of tryptophanyl and tyrosyl residues of proteins by second-derivative fluorescence spectroscopy

Second-derivative spectroscopy has been applied to the study of the fluorescence of aromatic amino acids. The spectral features of the second derivative emission spectra of free aromatic amino acids and proteins are described, the emission of each aromatic fluorophore being characterized by a particular minimum-maximum pair. An easy, accurate, and rapid method is proposed for the quantitative determination of tyrosine and tryptophan, based on the addition of small amounts of a standard solution to the samples followed by the measurement of the increase in the distance between a selected minimum and an adjacent maximum, in the second-derivative spectrum. For tyrosine determination, excitation wavelength was 275 nm, and the selected minimum-maximum (m,M) pair was (300; 330 nm), while an excitation of 300 nm and a minimum-maximum pair (357; 377 nm) were employed for the tryptophan determination. This method enables the tryptophan content of proteins to be determined directly, without the need for correction for the presence of tyrosine. The tyrosine content of proteins can also be determined at neutral pH, in the presence of both tryptophan and phenylalanine. The proposed method has also been applied to trypsin activation of frog epidermis tyrosinase.     

Effect of an induced conformational change on the physical properties of two chemotactic receptor molecules.

The physical properties and conformational dynamics of the Salmonella typhimurium ribose and galactose receptors have been examined. Studies involving circular dichroism, fluorescence, absorption spectroscopy, and sedimentation analysis show that the two receptor proteins have different morphologies and exhibit diverse responses to sugar binding. The ribose receptor lacks both tryptophan and disulfide residues, and the galactose receptor lacks disulfides and has only a single tryptophan residue. By virtue of these fortuitous properties, the conformational changes induced in these proteins by sugar binding can be dissected by utilizing a variety of physical probes. A ligand-induced conformational change in the ribose receptor is shown by circular dichroism and fluorescence spectroscopy, which reveal spectral changes assignable to tyrosine, phenylalanine, and methionine residues. A conformational change in the galactose receptor has been demonstrated by fluorescence spectroscopy involving the distant reporter group method, which shows changes assignable to tryptophan and methionine sites and which is corroborated by sedimentation analysis. It is clear that there are extensive conformational changes in the two receptor proteins and that the different physical methods provide complementary information on the nature of these changes.     

Large neutral amino acids: dietary effects on brain neurochemistry and function

The ingestion of large neutral amino acids (LNAA), notably tryptophan, tyrosine and the branched-chain amino acids (BCAA), modifies tryptophan and tyrosine uptake into brain and their conversion to serotonin and catecholamines, respectively. The particular effect reflects the competitive nature of the transporter for LNAA at the blood–brain barrier. For example, raising blood tryptophan or tyrosine levels raises their uptake into brain, while raising blood BCAA levels lowers tryptophan and tyrosine uptake; serotonin and catecholamine synthesis in brain parallel the tryptophan and tyrosine changes. By changing blood LNAA levels, the ingestion of particular proteins causes surprisingly large variations in brain tryptophan uptake and serotonin synthesis, with minimal effects on tyrosine uptake and catecholamine synthesis. Such variations elicit predictable effects on mood, cognition and hormone secretion (prolactin, cortisol). The ingestion of mixtures of LNAA, particularly BCAA, lowers brain tryptophan uptake and serotonin synthesis. Though argued to improve physical performance by reducing serotonin function, such effects are generally considered modest at best. However, BCAA ingestion also lowers tyrosine uptake, and dopamine synthesis in brain. Increasing dopamine function in brain improves performance, suggesting that BCAA may fail to increase performance because dopamine is reduced. Conceivably, BCAA administered with tyrosine could prevent the decline in dopamine, while still eliciting a drop in serotonin. Such an LNAA mixture might thus prove an effective enhancer of physical performance. The thoughtful development and application of dietary proteins and LNAA mixtures may thus produce treatments with predictable and useful functional effects.     

Physicochemical characterization of bovine retinal arrestin

The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 degrees C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.     

Examination of phenylalanine microenvironments in proteins by second-derivative absorption spectroscopy

We have employed near ultraviolet derivative absorption spectroscopy to study the microenvironments of phenylalanine residues in proteins. The use of second-derivative uv spectra in the 250- to 270-nm range effectively suppresses spectral contributions from tryptophan and tyrosine residues. Fitting a polynomial to the numerically calculated second-derivative spectrum allows precise determination of the position of the negative derivative peak near 258 nm. This position is shown to be correlated with the polarity of the microenvironments of phenylalanine residues. This approach allows monitoring of changes in the state of phenylalanine side chains during folding/unfolding of the proteins. In addition, this method permits perturbation of protein samples with ethylene glycol to be used to establish the relative degree of solvent exposure of protein phenylalanine.     

5-Hydroxytryptophan: An absorption and fluorescence probe which is a conservative replacement for [A14 tyrosine] in insulin

Use of insulin's intrinsic tyrosine absorption and fluorescence to monitor its interaction with the insulin receptor is limited because the spectral properties of the receptor tryptophan residues mask the spectral properties of the hormone tyrosine residues. We describe the synthesis of an insulin analog where A14 tyrosine is replaced by a tryptophan analog, 5-hydroxytryptophan. This insulin is spectrally enhanced since 5-hydroxytryptophan has an absorption band above 300 nm which is at lower energies than the absorption of tryptophan. Steady-state and time-resolved fluorescence parameters indicate that 5-hydroxytryptophan reports the same information about the environment of the A14 side chain as does the corresponding tryptophan-containing insulin. The synthetic hormone is a full agonist compared to porcine insulin, but has slightly reduced specific activity. Consequently, this spectrally enhanced insulin analog will be useful for hormone-receptor interaction studies since it can be observed by both absorption and fluorescence even in the presence of the tryptophan-containing receptor.     

Diffuse reflectance spectroscopy of fibrous proteins

UV-visible diffuse reflectance (DR) spectra of the fibrous proteins wool and feather keratin, silk fibroin and bovine skin collagen are presented. Natural wool contains much higher levels of visible chromophores across the whole visible range (700-400?nm) than the other proteins and only those above 450?nm are effectively removed by bleaching. Both oxidative and reductive bleaching are inefficient for removing yellow chromophores (450-400?nm absorbers) from wool. The DR spectra of the four UV-absorbing amino acids tryptophan, tyrosine, cystine and phenylalanine were recorded as finely ground powders. In contrast to their UV-visible spectra in aqueous solution where tryptophan and tyrosine are the major UV absorbing species, surprisingly the disulphide chromophore of solid cystine has the strongest UV absorbance measured using the DR remission function F(R)(∞). The DR spectra of unpigmented feather and wool keratin appear to be dominated by cystine absorption near 290?nm, whereas silk fibroin appears similar to tyrosine. Because cystine has a flat reflectance spectrum in the visible region from 700 to 400?nm and the powder therefore appears white, cystine absorption does not contribute to the cream colour of wool despite the high concentration of cystine residues near the cuticle surface. The disulphide absorption of solid L: -cystine in the DR spectrum at 290?nm is significantly red shifted by ~40?nm relative to its wavelength in solution, whereas homocystine and lipoic acid showed smaller red shifts of 20?nm. The large red shift observed for cystine and the large difference in intensity of absorption in its UV-visible and DR spectra may be due to differences in the dihedral angle between the crystalline solid and the solvated molecules in solution.     

Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

Quantitative studies in molecular and structural biology generally require accurate and precise determination of protein concentrations, preferably via a method that is both quick and straightforward to perform. The measurement of ultraviolet absorbance at 280 nm has proven especially useful, since the molar absorptivity (extinction coefficient) at 280 nm can be predicted directly from a protein sequence. This method, however, is only applicable to proteins that contain tryptophan or tyrosine residues. Absorbance at 205 nm, among other wavelengths, has been used as an alternative, although generally using absorptivity values that have to be uniquely calibrated for each protein, or otherwise only roughly estimated. Here, we propose and validate a method for predicting the molar absorptivity of a protein or peptide at 205 nm directly from its amino acid sequence, allowing one to accurately determine the concentrations of proteins that do not contain tyrosine or tryptophan residues. This method is simple to implement, requires no calibration, and should be suitable for a wide range of proteins and peptides.     

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.     

A simple colorimetric method for the determination of tryptophan

A simple, sensitive, and reproducible colorimetric method for the determination of tryptophan in amounts as low as 2 μg is described. It is based on the oxidation of tryptophan by sodium nitrite and the coupling of the oxidized product to the leucodye N-1-(naphthyl)ethylenediamine dihydrochloride. The purple-pink product has an absorption maximum at 550 nm. There is no interference by carbohydrates, other amino acids, neutral salts, or a number of other compounds likely to be found in tissue hydrolysates. A number of indole derivatives including indole-3-acetic acid also react to give a colored product. Dipeptides containing tryptophan are much less reactive than free tryptophan; hence proteins must be hydrolyzed completely for the method to be useful. The assay is carried out at room temperature and can be modified easily to increase or decrease its sensitivity. It has been employed to determine the tryptophan content of a number of proteins following alkaline hydrolysis. Generally, values obtained were in close agreement with values reported in the literature.     

Cytoprotective antioxidant function of tyrosine and tryptophan residues in transmembrane proteins.

The transmembrane domains of integral membrane proteins show an astounding accumulation of tyrosine and tryptophan residues, especially in the region of the highest lipid density. We found that these residues perform vital antioxidant functions inside lipid bilayers and protect cells from oxidative destruction. First, tyrosine- and tryptophan-containing peptides representing stretches from the transmembrane domains of different integral membrane proteins, including presenilin and the cystic fibrosis transmembrane conductance regulator, prevent oxidative lysis in clonal and primary cells. Second, long-chain acylated tyrosine and tryptophan, but not phenylalanine or short-chain acylated derivatives, are potent inhibitors of lipid peroxidation and oxidative cell death. The antioxidant functions of tyrosine and tryptophan may provide a specific explanation for (a) their unique transmembrane distribution pattern and (b) the high vulnerability of low-protein neuronal membranes to oxidative stress, as seen in neurodegenerative disorders.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

Photoacoustic spectroscopy of aromatic amino acids in proteins

This paper concerns the use of photoacoustic spectroscopy (PAS) to study the presence of aromatic amino acid in proteins. We examined the aromatic amino acids in six proteins with well-known structures using absorption spectra of near ultraviolet PAS over the wavelength range 240–320 nm. The fundamental understanding of the physical and chemical properties that govern the absorption of light and a subsequent release of heat to generate a transient pressure wave was used to test the concept of monitoring aromatic amino acids with this method. Second derivative spectroscopy in the ultraviolet region of proteins was also used to study the regions surrounding the aromatics and the percentage area in each band was related in order to determine the contribution in function of the respective molar extinction coefficients for each residue. Further investigation was conducted into the interaction between sodium dodecyl sulphate (SDS) and bothropstoxin-I (BthTx-I), with the purpose of identifying the aromatics that participate in the interaction. The clear changes in the second derivative and curve-fitting procedures suggest that initial SDS binding to the tryptophan located in the dimer interface and above 10 SDS an increased intensity between 260 and 320 nm, demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interactions. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scattering is significant.     

Spectroscopic characterization of the ATPase of the thermophilic bacterium PS3 and its isolated subunits

The ATPase of the thermophilic bacterium PS3, TF0F1, and its subunits has been isolated and their absorption and fluorescence spectra have been measured. The following results were obtained: The tryptophan content of the subunits was determined spectroscopically. Although tryptophan (Trp) and tyrosine (Tyr) are found in TF1, the fluorescence spectrum of native TF1 and its subunits is dominated by Tyr fluorescence; this is in contrast to other proteins. Among (native) TF1 and its subunits only TF1 and the alpha-subunit show a weak fluorescence of Trp, which is blue-shifted, indicating a location in a strongly hydrophobic environment. TF0 fluorescence is dominated by the strong Trp fluorescence. TF0F1 fluorescence is also dominated by the Trp residues. Additionally, its fluorescence is higher than the sum of the isolated TF0 and TF1, indicating marked changes in the microenvironment of the fluorescing aminoacids upon binding of TF1 to TF0.     

Structural Changes of Human Serum Albumin Induced by Cadmium Acetate

The structural changes of human serum albumin (HSA) induced by the addition of cadmium acetate were systematically investigated using UV–vis absorption, circular dichroism (CD), synchronous, and three‐dimentional (3D) fluorescence methods. The fluorescence spectra suggested the formation of cadmium acetate–HSA complex. UV absorption result indicated that the interaction between cadmium acetate and HSA could lead to the alteration of the protein skeleton. The structural analysis according to CD method showed that the cadmium acetate binding altered HSA conformation with a major reduction of α‐helix, inducing a partial protein unfolding. Synchronous fluorescence spectra suggested that cadmium acetate was situated closer to tryptophan residue compared to tyrosine residues, making tryptophan residue locate in a more hydrophobic environment. 3D fluorescence demonstrated that cadmium acetate could induce the HSA aggregation and cause a slight unfolding of the polypeptide backbone of the protein.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.